Development of an HIV vaccine has proven difficult; although recent studies suggest that some protection against HIV is possible. Over the past 16 years, antibodies have been found in HIV-infected (and HIV-uninfected) humans that neutralize diverse isolates of HIV in vitro. Administration of a cocktail of these broadly neutralizing HIV antibodies decreased HIV viral load in infected humans and to reduced HIV transmission in a monkey model of HIV transmission. Extensive characterization of these HIV neutralizing antibodies has been conducted, and it is known that the some of these antibodies are polyspecific, recognizing a hidden region on the HIV glycoprotein that is involved in virus-cell fusion (gp41), and with cellular constituents that may also play a role in HIV entry. Thus far, the major limitation of thee antibodies is that they have not been able to be reproducibly or potently produced following immunization with either the HIV gp41 or with cellular components. I found that a common, apparently nonpathogenic human virus (GB virus C) inhibits HIV replication in vitro, and that HIV infected people who are coinfected with GBV-C live longer than those who do not have GBV-C. We found that GBV-C infects lymphocytes, including CD4 cells, and others found that the GBV-C envelope glycoprotein E2 inhibits HIV fusion, much like a piece of the gp41 molecule. E2 peptides have been shown to inhibit fusion as well. These studies led me to hypothesize that antibodies to E2 might interfere with HIV entry, and studies described in this application show that a variety of GBV-C E2 antibodies neutralize HIV infectivity in vitro. Immunization of rabbits, mice, and monkeys with recombinant GBV-C E2 protein elicits antibodies that react with E2 protein, but also react with a cellular-based molecule on HIV particles. This cellular molecule is not exposed on the surface of cells. Thus we hypothesize that GBV-C E2 protein mimics the structure of a cellular molecule that is enriched on HIV particles, and that binding to this antige decreases HIV infectivity. The purpose of this proposal is to: 1) generate tools that will allow further characterization of the E2 and HIV particle molecule recognized by these antibodies, and determine the optimal immunization method for raising antibodies to GBV-C E2, and 2) characterize and identify the cellular molecule involved in E2 antibody mediated neutralization. These studies will provide insight into a new method of generating a HIV vaccine, and may provide new molecules that can be targeted for the development of HIV treatments. Since the VA provides medical care to the largest single population of HIV-infected people in the United States, this research is highly relevant to the VA research mission.